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Nanotechnology/Nano-bio Primer

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Bio-nanotechnology

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Biological 'units'

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Biosystem building blocks

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make up cell membranes and these are the very important barriers that control what can enter and exit a cell. The importance can be seen in the fact that one third of all proteins are membrane proteins.

Cell Energy Supply and Consumption

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The cell organelle the mitochondria is the power plant of the cell energy metabolism.

It synthesises ATP, the basic energy source for numerous processes in cells. The synthesis proces is driven by an electric potential (~150mV) across the mithocondrial inner membrane that is maintained by ion pumps in the membrane that make the outside more acidic while the inner matrix region is more alkaline. The gradient is ~0.5 pH units [1]. The membrane potential is not homogeneous over the mitochondria [2]

The Chemiosmotic theory of the ATP synthesis was developed by Peter Mitchell in 1961, who was later rewarded the chemistry Nobel prize for it in 1978.

Current research on Mitocondria was reviewed eg. in Science 28 aug 1998 and 5 mar 1999.

The membrane potential difference is small, but the menbrane is also very thin; approx 5-7nm, giving an electrical field across the membrane of the order 30MV/m - which will make any physicist designing high energy particle accelerators quite envious - the value is huge compared to electrical fields that would make large scale matter break down. Its is on contrast to the otherwise weak forces normally encountered when talking about the physical properties of lipid membranes. Despite being a thin and soft membrane that can easily be ruptured by mechanical contact, it is capable of withstanding extreme electrical fields.

The membrane potential can be observed by using the fluorescent probe JC-1 -or more verbosely 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide. JC-1 is a lipophilic cation that can penetrate the (lipo)membrane of the cell and mitochondria. JC-1 can be excited by green laser light and emit fluorescent green light at 530nm when it is in a monomeric form, but if the membrane potential is increased above a threshold around 80-100mV, JC-1 will aggregate and the fluorescense becomes increasingly orange at 590nm [3]. JC-1 is a very tested and reliable marker for the membrane potential [4] [5]. A recipe for the use of JC-1 can be found in [6].

The immunesystem

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Fluorescent Markers

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Natural cells and biological structures often have very little contrast when seen in optical microscopes. Take a normal yeast cell and look at it under a microscope and you will only see small balls with no structure. Fluorescent markers and dyes can be used to stain specific substances inside the cell which would otherwise not give an appreciable optical signal. Such markers have for many years been essential to get proper images of biological samples. No matter what type of microscope is used, the fluorescent markers are widely used to enhance the contrast and signal to noise ratio in measurements.

Using fluorescent dyes and recording spectra of light emitted from whole or selected parts of cells can give valuable information [7], such as:

  • The functional and structural characteristics of normal or malignant cells
  • The intracellular dynamics of molecules that are naturally occurring, or added to the cell such as drugs.
  • Characterization of the interactions between cells, or the cell and its surrounding media.
  • Intracellular dynamics of ions such as Ca++, Mg++, or other important variables such as pH or membrane potentials by using fluorescent markers for the chemicals and potentials under investigation.

The fluorescent markers are used for many other techniques than just microscopy. A method called flow cytometry is very efficient for analyzing large numbers of individual cells. One by one, individual cells pass through a thin channel where they are exposed to the exciting laser light and the emitted fluorescense and absorption is detected by light sensors. The technique gives very good statistical results, but does not allow the detection of eg. individual mithocondria inside a well functioning cell but will rather give an average value of the state of all mithocondria in a single cell.

Ressources

Lengths and Masses in biochemistry

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1Da is one atomic mass unit and approximately the mass of a proton. It is about one thousandth of a zeptogram = 10^-24g

Sizes of small life forms

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  • Nanobes Nanobes are tiny filamental structures first found in some rocks and sediments. from 20 nm.
  • Parvovirus - a family of viruses down to 20 nm
  • 'Nanobacteria' or 'Calicifying Nanoparticles (CNP)' - a recently discovered class of nanoparticles that seem related to various calcification processes in the body and may be living [see also new scientist 23 june 2007 p38]. 50-100nm
  • Nanoscale microbes (Nanoarchaeota a kind of archaea) [science vol 314 p1933]
  • Smallest bacterium ' mycoplasma genitalium' -M. genitalium was also considered to be the organism with the smallest genome. 300 nm
  • Nanoarchaeum equitans a thermophile 400 nm
  • The largest virus 'mimivirus', that infects amoebaes 400nm
  • Possibly the most abundant organism on earth, the marine bacterium 'pelagibacter ubique (SAR11)' 500 nm
  • Typical gut bacteria 'E. Coli' 2000-6000 nm.

References

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See also notes on editing this book about how to add references Nanotechnology/About#How_to_contribute.

  1. ↑ Mitochondrial diseases in man and mouse, Wallace DC, Science, vol. 283 (5407): 1482-1488 MAR 5 1999.
  2. ↑ Intracellular Heterogeneity In Mitochondrial-Membrane Potentials Revealed By A J-Aggregate-Forming Lipophilic Cation Jc-1, Smiley St, Reers M, Mottolahartshorn C, Lin M, Chen A, Smith Tw, Steele Gd, Chen Lb, Proceedings Of The National Academy Of Sciences Of The United States Of America, Vol. 88 (9): 3671-3675 May 1991 Letters, vol. 78 (11): 1637-1639 MAR 12 2001.
  3. ↑ Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1, Andrea Cossarizza and Stefano Salvioli, Purdue Cytometry CD-ROM Series,volume 4[1].
  4. ↑ Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes, Mathur A, Hong Y, Kemp BK, Barrientos AA, Erusalimsky JD, Cardiovascular Research, vol. 46 (1): 126-138 APR 2000
  5. ↑ JC-1, but not DiOC(6)(3) or rhodamine 123, is a reliable fluorescent probe to assess Delta Psi changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis, Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A, FEBS Letters, vol. 411 (1): 77-82 JUL 7 1997
  6. ↑ Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1, Andrea Cossarizza and Stefano Salvioli, Purdue Cytometry CD-ROM Series,volume 4[2].
  7. ↑ Manfaits webpage on Le Groupement De Recherche 1860 at the Centre National de la recherche scientifique, [3]
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